Histone-histone interaction mediates chromatin unfolding at physiological ionic strength

High-resolution thermal denaturation data on chicken erythrocyte chromatin are reported over 4 orders of magnitude in NaCl concentration which includes the physiological region. A novel technique using critical-point polyacrylamide sols instead of ordinary solvents effectively stabilizes chromatin against precipitation at high salt concentrations. These sols are optically transparent from 260 to 320 nm and are thermally stable over the temperature ranges studied. At Na+ ion concentrations below 10 mM, the polyacrylamide slightly destabilizes chromatin at the nucleosome level, possibly through interactions of histones H1 and H5 with the carboxylic acid residues. At the same low salts, polyacrylamide stabilizes pure DNA against denaturation, presumably by mechanically stabilizing it against helix-distorting thermal fluctuations. In both cases, however, the polyacrylamide sols are entirely noninvasive at higher salts. Prominent low-temperature thermal transitions are observed in chromatin at and above 100 mM NaCl which evidently are associated with conformational changes in DNA. Our results are in accord with the idea that histone-histone interactions at physiological ionic strengths (approximately 100 mM Na+) may be comparable to histone-DNA interactions and hence may be sufficient to promote the destabilization of the DNA helix in chromatin under these conditions. The biological implications of this are discussed, and a possible model for the local decondensation of chromatin under physiological conditions is proposed.     

Mechanical properties of a reversible, DNA-crosslinked polyacrylamide hydrogel

Mechanical properties of a polyacrylamide gel with reversible DNA crosslinks are presented. In this system, three DNA strands replace traditional chemical crosslinkers. In contrast to thermoset chemically crosslinked polyacrylamide, the new hydrogel is thermoreversible; crosslink dissociation without the addition of heat is also feasible by introducing a specific removal DNA strand. This hydrogel is characterized by a critical crosslink concentration at which gelation occurs. Below the critical point, a characteristic temperature exists at which a transition in viscosity is observed. Both temperature-dependent viscosity and elastic modulus of the material are functions of crosslink density.     

The Presence of Novel Plant Growth Regulators in Leaves of Distylium racemosum Sieb et Zucc

The scaling law derived from the percolation theory was applied to the concentration dependence of mechanical properties of polyacrylamide measured near the sol–gel transition point. The critical concentration of the sol–gel transition, ?_g, was estimated from the plot of concentration (?) vs. the reciprocal of viscosity (η) by extrapolating 1/η to zero. The critical exponent for the sol viscosity, s, which was estimated from the slope of the log(?_g–?) vs. log η plot was about 0.7. The estimated value of s was similar to the value predicted by the percolation theory based on the superconductor–normal conductor mixture model. The critical exponent for the gel elasticity, t, as estimated from the slope of the log(?–?_g) vs. log G′ plot, where G′ was the dynamic shear modulus of the gel at a frequency of 2Hz. The value of t was about 2, which was also similar to the value predicted by the percolation theory. These results indicated the at the concentration dependences of η and G′ of polyacrylamide near the sol–gel transition point were described by the percolation theory.     

Sol–sol structural transition of aqueous agarose systems

A structural transition is reported to occur in aqueous sols of agarose, an electrically uncharged biostructural polysaccharide. The transition has no measurable effect on size dispersity on the shape of the solute polysaccharide as observed by precision photon correlation spectroscopy. It originates a low-angle pattern of scattered light similar to that which monitors phase separations in polymer blends. Thus, it must be due to some extent to spatially modulated polymer clustering, typical of spinodal decomposition. In the interval of temperatures studied, it precedes very distinctly in time the thermoreversible sol–gel transition, which is known to be promoted at higher concentrations. It also anticipates to an appreciable extent the spatial density modulation observed in the gel. Although reported here for the first time, a spinodal decomposition of the sol that precedes and possibly triggers the processes leading to gelation does not come unexpectedly in terms of site-bond correlated-percolation theory. In general, this occurrence raises the question as to whether the spontaneous onset of regions of higher and lower polymer concentration (spinodal separation) may be regarded as a novel path for biomolecular interactions and the self-assembly of order in biomolecular systems.     

Purification and properties of a thiol protease from rat liver nuclei

A thiol protease was purified about 800-fold from the chromatin fraction of rat liver by employing Sepharose 6B gel filtration, chromatofocusing and Sephadex G-100 gel filtration. It was nearly homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and its molecular weight was about 29000. The isoelectric point of the enzyme was 7.1. The pH optimum for degradation of 3H-labelled ribosomal proteins was 4.5. It is noticeable that the maximal activity was shifted to pH 5.5 by DNA, and that 30-40% of the maximal activity was observed at neutral pH in the presence of DNA. The activity was increased about twice by 2-4 mM dithiothreitol. The protease may be specific for the nuclei because it is different from all lysosomal thiol proteases ever known.     

DNA cleavage in chromatin spacer segments by a non-enzymic probe, 1,10-phenanthroline-copper complex

The action of 1,10-phenanthroline-copper complex under aerobic conditions in the presence of 2-mercaptoethanol on chromatin DNA in murine thymocyte nuclei was studied. At limited oxygen supply primarily multiple single-strand breaks of DNA in the spacer segments are observed while the core DNA segments in chromatin remain intact. After the single-strand breaks accumulate in both DNA strands (under conditions of improved oxygen supply), double-strand cleavage of DNA to fragments of nucleosomal size becomes apparent. This regular character of DNA degradation in chromatin is apparently due to the preferential binding of the 1,10-phenanthroline-copper complex to DNA of the spacer segments and to the localized generation of damaging radicals.Abbreviations OP 1,10-phenanthroline - OP-Cu 1,10-phenanthroline-copper complex - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulphate - 2-ME 2-mercaptoethanol     

Analysis of chromatin of the brain of young and old rats by micrococcal nuclease and DNase I

Micrococcal nuclease (MCN) and DNase I were used to study the conformational changes in chromatin of the brain of rats of different ages. Purified nuclei and chromatin were digested separately by MCN and DNase I. Kinetics of digestion of chromatin by MCN are similar for young, adult and old rats. Also agarose gel electrophoresis of DNA fragments do not show any differences. The kinetics of digestion with DNase I, on the other hand, are greater and faster for 20-week old rats than for 90-week old rats. High performance denaturing polyacrylamide gel electrophoresis reveals that a greater amount of smaller fragments of DNA are produced in the 20-week old rats than in the 90-week. These conformational changes occur in the chromatin during aging.     

Tissue specificity of nonhistone proteins from human chromatin

Summary Nonhistone proteins were isolated from human placental and tonsillar chromatins. Antiserum was prepared against a complex from some nonhistone proteins and DNA (NP-DNA) from placental chromatin. With the help of polyacrylamide gel electrophoresis and immunological methods the tissue specificity of human chromatin nonhistone proteins was established. The described organ immunogenic specificity of the complex of DNA and nonhistone proteins (NP-DNA) from human chromatin is in accordance with data published on similar complexes from different animal organs. Besides, it is shown that shearing of chromatin leads to large chifts in NP-DNA concentrations required for maximum complement fixation in the presence of the prepared antiserum. This may probably be due to a damage of certain chromatin super structures which involve some of the nonhistone proteins and DNA sequences from both the more condensed and less condensed parts of chromatin.     

Fractionation of L-cell chromatin into DNA, RNA, and protein fractions on Cs2SO4 equilibruim density gradients

A new procedure is described for fractionation of chromatin into DNA, RNA, and total chromatin protein. By isopycnic gradient centrifugation of chromatin preparations in Cs₂SO₄ solutions containing dimethylsulfoxide and sodium sarcosyl it is possible to obtain highly purified fractions of these components. The method gives a very high yield of these chromatin fractions unlike some other methods, where irreversible binding to columns occurs. Also with this method it is possible to obtain highly concentrated fractions, which after a simple dialysis step, can be conveniently analyzed by polyacrylamide gel electrophoresis.Nuclei from L-929 cells were isolated by a method involving citric acid or by a method using a nonionic detergent. The yields of DNA obtained by both methods was compared. Chromatin was isolated from purified nuclei (prepared in either of the above ways) in two different ways also. In one method, chromatin was extracted from nuclei with 1 m NaCl. A second method involving fractionation of lysed nuclei in sucrose and metrizamide solutions was also used. The yields of DNA obtained by both methods was compared. There appears to be little nuclear membrane contamination of any of these chromatin preparations.A preliminary analysis of L-929 cell chromatin total RNA and protein fractions on polyacrylamide and agarose gels has been made. Both fractions appear to be quite complex with a wide spectrum of subcomponents of differing S values.     

Solubilization of Jerusalem artichoke tuber chromatin by 2,4-dichlorophenoxyacetic acid

Chromatin from the tuber of the Jerusalem artichoke (Helianthus tuberosus) was solubilized in 2,4-dichlorophenoxyacetic acid (2,4-D) solution (100 mM) at pH 7.0. This solubilization was much affected by the pH; below 6.0 less chromatin was solubilized. The elution pattern of the products on gel filtration with Sepharose 4B showed that the solubilization was caused by the dissociation of the DNA and associated proteins. The pattern of polyacrylamide gel electrophoresis of histones extracted from the chromatin solubilized by 2,4-D was quite different from those of histones extracted from the original chromatin or from NaCl (2.0M) solubilized chromatin. The F1 and F3 fractions seemed to be little affected by 2,4-D, but the F2a1, F2a2 and F2b fractions were greatly decreased. In addition, the ratios of histones and non-histone proteins to DNA changed considerably in 2,4-D solubilized chromatin in an inverse manner. None of these changes were observed with NaCl. which suggests that the behaviour of 2,4-D for the solubilization of chromatin differs substantially from that of NaCl.     

DNA-relaxing enzyme from Micrococcus luteus.

A DNA-relaxing enzyme which catalyzes the conversion of superhelical DNA to a non-superhelical covalently closed form has been purified from Micrococcus luteus to near homogeneity by two chromatographic steps. The enzyme is a single polypeptide chain. As determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel filtration on Sephadex G 150, the molecular weight is 115,000. The DNA-relaxing activity determined as a function of enzyme concentration follows a sigmoidal curve. The enzyme requires Mg++ for activity. In the presence of 4.5 mM Mg++ addition of 50-250 mM KCl yields incompletely relaxed DNA molecules (intermediates); intermediates are also observed in the absence of KCl, when the reaction is carried out at 0 degree C or at Mg++ concentrations exceeding 10 mM.     

IMPROVEMENTS IN THE ION-EXCHANGE METHOD OF PREPARING SILICA SOLS

SUMMARY: An improved ion-exchange column for producing stable silica sols is described. Best results were obtained with a sulphonic-polystyrene resin. The storage life of sols so produced may be extended to as much as two months by refrigeration and acidification with hydrochloric acid. Silica sol has a buffering action in the region of pH 2. Silica gel media prepared from such sols are comparable in cost with agar media.     

Non-histone protein synthesis during G1 phase and its relation to DNA replication.

The kinetics of non-histone chromosomal protein (NHCP) synthesis were studied in Chinese hamster ovary (CHO) plateau phase cells stimulated to proliferate and were compared to NHCP synthesis kinetics in two populations of synchronous G1 traversing cells. In all cases, NHCP synthesis rates increase 3- to 5-fold as cells traversed G1 and attained maximum values one hour before semi-conservative DNA replication began. Similar to results in synchronous G1 cells, the molecular weight distributions of the NHCP fraction from stimulated plateau phase cells underwent only minor changes, measured by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis, as these cells moved toward S phase. Yet, during this progression after plateau phase and in the transition from early G1 to late G1 in synchronous cells, the total NHCP fraction increased significantly (1.5-2-fold) in amount per cell. These data indicate that plateau phase cells are similar to early G1 cells both in terms of their amounts of non-histone per cell and in their subsequent NHCP synthesis kinetics as they move toward S phase. These results extend previous findings which suggested that NHCP synthesis was coupled to DNA replication and demonstrate that the increased NHCP synthesis and accumulation in chromatin may be a biochemical marker for G1 progression.     

Histones of Neurospora crassa.

Neurospora crassa chromatin isolated by a rapid method minimizing proteolytic degradation contains approximately one weight of acid-extractable basic protein per weight of DNA. This basic protein consists of five major polypeptide species which are similar in size to the histone proteins of higher eukaryotes and are present in approximately the same molar ratios. These five polypeptides have been purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Their electrophoretic mobilities in polyacrylamide gels and their amino acid compositions indicate that they are histones homologous, although not identical, to the H1, H2A, H2B, H3, and H4 histones of mammals. The first 3 residues in the amino acid sequence of Neurospora H3 histone are identical to the first 3 residues in calf and pea H3; Neurospora H1, H2A, and H4 histones have blocked NH2 termini, like their mammalian counterparts. The finding of recognizable H1, H2A, H2B, H3, and H4 histones in Neurospora extends the range of eukaryotes now shown to contain a full complement of these strongly conserved chromosomal proteins, and supports the view that histones became involved in chromosome structure at a very early point in the evolution of eukaryotes.     

Identification of nonhistone chromatin proteins in chromatin subunits (or mononucleosomes) devoid of histone H1.

Rat liver chromatin was digested by micrococcal nuclease. Chromatin subunits (or mononucleosomes) were isolated by sucrose density gradient and subsequently fractionated by 6% polyacrylamide gel electrophoresis into two major components. One component (MN1) of the mononucleosomes had a higher mobility, contained histones H2A, H2B, H3, H4, and shorter DNA fragments (140 base pairs) while the other (MN2) contained all five histones and longer DNA fragments (180 base pairs). Both submononucleosomes (MN1 and MN2) were found to contain nonhistone chromatin proteins (NHCP). By electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel, 9 and 11 major fractions of NHCP were identified in the submononucleosomes MN1 and MN2, respectively. It was also observed that treatment of mononucleosomes with 0.6 M NaCl removes most of these NHCP and histone H1 except for two major NHCP which remain in the core particles.     

Temporal synthesis and intranuclear accumulation of the nuclear acidic proteins during periods of chromatin reactivation in Physarum polycephalum

In the lower eukaryote Physarum polycephalum, depending upon the existing cell state (i.e., actively growing plasmodia or metabolically quiescent cysts), there is in the complement of acidic chromatin proteins certain “proliferation” or “nonproliferation” associated proteins. Nonproliferative microplasmodia can be induced to undergo a 12 hr period of physiological and metabolic reorganization resulting in mitosis, DNA synthesis, and the reestablishment of active synchronous growth. During the 12 hr period of chromatin reactivation the specific acidic proteins associated with inactive chromatin and nonproliferative cell states decrease in intranuclear concentration in a continuous and linear fashion. The specific proteins associated with metabolically active chromatin and proliferative cell states are synthesized preferentially at different times during the 12 hr transition period. While several of the proliferation-associated proteins increase continuously in intranuclear concentration during the reactivation period others show maximum increases in their intranuclear concentration during the 2 hr period just preceding mitosis and DNA synthesis. The changes which develop in the acidic chromatin and nucleolar proteins during the period of chromatin reactivation occur independent of and prior to DNA synthesis and mitosis. Incorporation studies using [¹⁴C]-glutamic acid have provided additional evidence for periods of pooled protein and delayed intranuclear binding. The relative specific activities of individual protein bands determined through gel radiography show temporal differences independent of intranuclear protein concentration.It has been estimated that the proteins which are synthesized and accumulate in the nucleus during periods of chromatin reactivation are present in intranuclear concentrations between 80,000 and 1,000,000 copies per nucleus.     

The Drosophila nuclear lamina protein YA binds to DNA and histone H2B with four domains

Dramatic changes occur in nuclear organization and function during the critical developmental transition from meiosis to mitosis. The Drosophila nuclear lamina protein YA binds to chromatin and is uniquely required for this transition. In this study, we dissected YA's binding to chromatin. We found that YA can bind to chromatin directly and specifically. It binds to DNA but not RNA, with a preference for double-stranded DNA (linear or supercoiled) over single-stranded DNA. It also binds to histone H2B. YA's binding to DNA and histone H2B is mediated by four domains distributed along the length of the YA molecule. A model for YA function at the end of Drosophila female meiosis is proposed.     

The instantaneous monitoring of polyacrylamide gels during electrophoresis.

The advantages of being able to see protein zones in a gel during electrophoresis (and hence before staining) are pointed out, and a method is described which depends on local increments of refractive index in these zones. The use of local increments of refractive index in polyacrylamide gels for measuring protein concentrations in zones during electrophoresis is briefly considered; it is found that such increments are greater than would be expected from the amount of protein when sodium dodecyl sulphate is present. The enhancement depends on conditions and time of running. This makes quantitative estimates difficult, but the sensitivity of detection of protein zones by observations based on refractive-index changes is greatly increased by this property of sodium dodecyl sulphate. Methods are described for making optically uniform gels (both with uniform and with graded concentrations of polyacrylamide), necessary for observation of small changes in refractive index. A simple dark-field system of observation is described. Examples are given showing protein samples observed with the system during electrophoresis and compared with the same gel stained with Coomassie Blue after completion of the run. Under optimal conditions the optical method is comparable in sensitivity with staining. With the proteins of lower mol.wt. (approx. 15000), the optical method is not so sensitive, becoming less sensitive with longer running time. This loss of sensitivity is greatly decreased by using more concentrated polyacrylamide gels, and graded gels are therefore more suitable for optical observation than are uniform gels. The observation of protein zones during electrophoresis adds nothing to the time needed for making a stained gel and gives much information long before it can be obtained from the stained gel.     

Effects of histone acetylation on nucleosome properties as evaluated by polyacrylamide gel electrophoresis and hydroxylapatite dissociation chromatography

The release of acetylated histones from chick oviduct chromatin was analyzed by hydroxylapatite column chromatography. By raising of the NaCl concentration, acetylated histones were eluted from hydroxylapatite-bound chromatin depending on their release from nucleosomal DNA. Electrophoresis on acid-urea gel showed that hyperacetylated forms of histone H4 were eluted at a lower NaCl concentration than non-acetylated or hypoacetylated H4, suggesting that hyperacetylated H4 has decreased stability in nucleosomes. However, under milder ionic conditions which do not induce dissociation between histones and DNA, polyacrylamide gel electrophoresis of purified nucleosome cores showed no evidence for their unfolding or for increased accessibility by high mobility group protein-17.     

Rheological study into the ageing process of high methoxyl pectin/sucrose aqueous gels

The ageing process of high methoxyl pectin (HMP)/sucrose gels was followed at different ageing temperatures by small amplitude oscillatory experiments. Dynamic mechanical measurements allowed the characterisation of the point at which the system undergoes the sol/gel transition. The HMP/sucrose system is extremely sensitive to temperature variation during ageing, especially in the lower temperature range. The viscoelastic behaviour through the gel point changes with the ageing temperature, probably due to variations in mobility of the pectin chains, and consequently, in the lifetime of junction zones. Weaker pectin networks are formed under thermal conditions unfavourable to the development of hydrophobic interactions. Gel time and elastic modulus have a complex dependence on temperature, which could be attributed to the different thermal behaviour of the intermolecular interactions that stabilise the nonpermanent cross links of these physical networks.